The invention relates to spherical, magnetizable polyvinyl alcohol microparticles which are suited in particular for use in diagnostic detection methods or for isolation and purification of biomolecules. The invention further relates to methods of production by means of which the said microparticles can be produced on a larger scale.
Magnetic particles of the above-mentioned type are being used in a large number of diagnostic methods or in biomedical or biomolecular research. Generally, they are spherical particles which contain a superparamagnetic material in colloidal form, said material being embedded in a polymer matrix or enveloped in a polymer shell. Typically, these magnetic particles are microparticles of a size in the range of 1 to 20 μm.
Due to their ability to bind target substances such as biomolecules or cells selectively and, as the case may be, reversibly, magnetizable microparticles are particularly suitable for use in automated processes. As the magnetizable microparticles can be temporarily immobilized by applying a magnetic field, separation from liquid media does not require any centrifugation steps. This facilitates automation even in multi-stage processes, and it leads to considerable savings in time.
Magnetizable polymer-based microparticles, their preparation and their use have already been described in the prior art, for example in WO 97/04862 A1 and in the printed publications cited therein.
WO 97/04862 A1 discloses bead-shaped or spherical particles of polyvinyl alcohol which are described therein as being suitable for the fractionation of cells, nucleic acids, proteins, viruses or bacteria, as well as for use in immunoassays, for DNA sequencing or DNA synthesis.
The particles described in WO 97/04862 A1 comprise a polymer matrix of polyvinyl alcohol which has magnetic colloids of particle sizes of 10-200 nm encapsulated therein. The bead-shaped or spherical polymer particles have a particle size in the range of 1-10 μm, preferably 1-4 μm.
The production of the magnetizable polymer particles described in WO 97/04862 A1 is executed in such a manner that an aqueous polyvinyl alcohol solution in which a magnetic colloid is dispersed is suspended at room temperature, by stirring, in an organic phase (e.g. vegetable oil) immiscible with the polymer phase and containing at least two emulsifying agents. During this suspension process, the polyvinyl alcohol is crosslinked by addition of a water-soluble crosslinking agent reacting with hydroxyl groups, for example glutaraldehyde. The polyvinyl alcohol particles can subsequently be modified for the specific binding of biomolecules, for example by grafting on of spacer molecules which can serve to bind biomolecules.
It has, however, turned out that methods of production described in WO 97/04862A1 and the particles obtainable thereby are disadvantageous in various respects.
First of all, it is problematic that the magnetizable polymer particles prepared in accordance with that known method have a too wide particle size distribution and that the particle size distribution can vary from one batch to another, that is, batch reproducibility is dissatisfactory. When using the particles in separation processes, these varying values lead to non-uniform yields so that in many applications these particles cannot be used.
In addition, the magnetizable polymer particles prepared in accordance with the method described in WO 97/04862 A1 contain high proportions of particles having a size of less than 0.5 μm and having a size of more than 3 μm.
Particles of a size of less than 0.5 μm do not have sufficient separation velocity, or mobility, in an external magnetic field, so that given the commonly used magnetic field strengths, they are separated only very slowly, which adversely affects the total processing time. In addition, there is a risk of these particles, which can be separated only slowly or not at all, to be carried over as impurities and impede and falsify subsequent reactions or measurements, e.g. UV measurements or polymerase chain reactions (PCR).
Particles of a size of more than 3 μm have the disadvantage of sedimenting relatively quickly in the gravitational field, which markedly limits their capacity of binding biomolecules. Hence, it can become necessary to counteract sedimentation by appropriate measures (re-dispersion). In addition, the greater the particle size of the magnetizable polymer particles, the smaller the total surface area, relative to the total mass or the suspension volume. This in turn leads to a lower yield in substances to be separated (biomolecules, cells). All in all, both the presence of particles that are too small (<0.5 μm) and the presence of particles that are too large (>3 μm) render the process of magnetic separation more difficult, for example in automated nucleic acid purification.
Furthermore, the magnetizable polyvinyl alcohol particles produced with the methods described in WO 97/04862 A1 contain only a relatively low content of magnetizable material (magnetite/iron oxide), namely in the range of about 7 to 24 percent by weight. This leads to insufficient or unfavorable separation properties of the particles in the magnetic field.
Owing to the above-described disadvantages in terms of particle sizes, particle size distribution and magnetite content, the usability of the magnetizable polymer particles prepared in accordance with the method described in WO 97/04862 A1 is limited. The magnetizable polymer particles are poorly suited, in particular, for use in automated separation methods and analytical methods.
It has turned out to be particularly disadvantageous that when used in PCR methods the magnetizable polymer particles known in the state of the art have an inhibitory effect, whereby the detection sensitivity is reduced and the accuracy and, thereby, reliability of the measurements are adversely affected. Since PCR techniques are widely used in molecular biology research and medical diagnostics, and the use of magnetizable microparticles is gaining in importance with regard to the automation of these methods, the above mentioned inhibiting effect of the particles known in the state of the art is highly dissatisfactory.
It has furthermore turned out that the methods of production described in WO 97/04862 A1 are not suitable for the production of magnetizable polyvinyl alcohol particles on a larger scale. With these known methods, the volume of the reaction batch is limited to a maximum of around 5 L, and the yield of magnetizable polymer particles is too low. Hence, it is not possible with these known production methods to cost-effectively produce magnetizable polyvinyl alcohol particles in larger quantities and with the required quality characteristics (particularly if the particle size distribution is narrow).